Mycoplasmas are small wall-less bacteria, primarily isolated from animal sources including humans. There are over 70 members of the genus Mycoplasma, and several related genera which are also characterized by small wall-less bacteria; these are Spiroplasma, Acholeplasma, Ureaplasma, Anaeroplasma, and Asteroleplasma. Only a handful of the species within these genera have been found associated with humans--some presumed to be "normal flora", others suspected of being pathogenic, and only one species known to be an important cause of human morbidity, whenever it is isolated. Mycoplasma pneumoniae is an important cause of primary atypical pneumonia and several nonrespiratory complications. The indication of its presence always provides clinically relevant information. Other pathogenic mycoplasma organisms comprise Mycoplasma fermentans, Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma genitalium. Nucleic acid compositions and methods for the detection of these organisms are the subject of two concurrently filed applications U.S. Ser. No. 07/673,661 and U.S. Ser. No. 07/673,687, now abandoned, entitled "Nucleic Acid Probes For The Detection of Genital Mycoplasmas" and "Nucleic Acid Probes For The Detection of Mycoplasma Fermentans Or The Aids-Associated Virus-Like Infectious Agent." At least one inventor is common to both of these applications and the present application.
The mycoplasmas, such as Mycoplasma pneumoniae, are fastidious organisms, requiring complex culture media containing peptone, yeast extract, expensive animal sera, and a sterol. Growth is relatively slow and reaches low cell densities compared to most bacteria. In addition, cell growth requires the addition of carbon dioxide to the surrounding atmosphere. For these reasons, many clinical laboratories are unable to perform culture isolation of M. pneumoniae, and consequently are left with no real ability to diagnose the presence of this important pathogenic bacteria. Given that mycoplasmas lack cell walls, antibiotics that target the bacterial cell wall, such as penicillin, have no anti-mycoplasma activity. Consequently, it is of importance for a physician to make a diagnosis of atypical pneumonia and prescribe the appropriate antibiotic.
Several investigators have discussed the similarity of Mycoplasma pneumoniae to Mycoplasma genitalium both serologically (Lind, et al., J. Clinical Microbiol., vol. 20, 1984) and within DNA sequences including the rRNA operons (Yogev and Razin, Int. Jnl. System. Bacteriol., vol. 36, 1986). Weisburg, et al. discuss the various other evolutionary relatives of M. pneumoniae (Jnl. of Bacteriol., vol. 171, 1989).
M. genitalium may possibly have a role in respiratory infection either in co-culture with M. pneumoniae or in the absence of M. pneumoniae (Tully, Clinical Micro. Newsletter, vol. 11, 1989). Mycoplasma genitalium may be responsible for some fraction of clinical atypical pneumonia.
While Kohne et al. (Biophysical Journal 8:1104-1118, 1968) discuss one method for preparing probes to rRNA sequences, they do not provide the teaching necessary to make probes to detect Mycoplasma pneumoniae or M. pneumoniae in combination with M. genitalium.
Pace and Campbell (Journal of Bacteriology 107:543-547, 1971) discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating such homology levels. Similarly, Sogin, Sogin and Woese (Journal of Molecular Evolution 1:173-184, 1972) discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships. Fox, Pechman and Woese (International Journal of Systematic Bacteriology 27:44-57, 1977) discuss the comparative cataloging of 16S ribosomal RNAs as an approach to prokaryotic systematics.
Hogan, et al. (International Patent Application, Publication Number WO 88/03957) describe five putative M. pneumoniae specific probes, but they were not tested in a manner which allowed evaluation of M. genitalium cross-reactivity by rigorous criteria. Their mixture of four probes reacted 10 times more strongly with M. genitalium than with the related species. M. gallisepticum. None of their probes target the 23S rRNA molecule. Probes disclosed appear to be unable to distinguish a known homologous sequence from M. genitalium.
Zivin and Monahan, European Patent application publication number 0305145A2, describe several probes which were designed based on the determination of 307 bases of the 5' end of M. pneumoniae. They claim anecdotally to exclude M. genitalium with some of their probes.
Gobel, et al. discuss probes for Mycoplasmas including M. pneumoniae (Gobel, et.al., Israel Jnl. of Med. Sci. vol. 23, 1987) but fail to teach what the structure of these probes is. Further, in an EP patent application number, EP0250662A1, Gobel et al. suggest probes to M. pneumoniae; however, such probes may lack the sensitivity or specificity for clinical applications and are not suitable for solution hybridization formats.
Rogers, et al. discuss sequences of 5S rRNA of mycoplasmas (Rogers et al., Proc. Natl. Acad. Sci. USA, vol. 82, 1985). Rogers, et al. do not suggest any sequences which would be useful to facilitate detection of these organisms and do not discuss 16S or 23S rRNA sequences. Woese, et al. discuss 16S rRNA oligonucleotide catalogs of selected mycoplasma species, but do not discuss probes or Mycoplasma pneumoniae.
Bernet, et al. discuss polymerase chain reaction detection of Mycoplasma pneumoniae (Bernet, et al., Jnl. Clin. Micro., vol. 27, 1989), but not directed toward rRNA or rDNA sequences. Hyman, et al. also discuss polymerase chain reaction detection of Mycoplasma pneumoniae and Mycoplasma genitalium (Hyman, et al., Jnl. Clin. Micro., vol. 25, 1987), but fail to discuss the utility of rRNA or rDNA sequences.
Gobel, et al. discuss mycoplasma rRNA probes in a publication, (Gobel, et al., Jnl. Genl. Micro., vol 133, 1987). However, Gobel, et al. do not recognize the significance of discrimination of probes toward other mycoplasma species, particularly Mycoplasma genitalium.
Ribosomes are of profound importance to all organisms because they serve as the only known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Bacterial ribosomes contain three distinct RNA molecules which, at least in Escherichia coli, are referred to as 5S, 16S and 23S rRNAs. In eukaryotic organisms, there are four distinct rRNA species, generally referred to as 5S, 18S, 28S, and 5.8S. These names historically are related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, ribosomal RNA molecules vary substantially in size between organisms. Nonetheless, 5S, 16S, and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacterium, including the mycoplasmas, and this convention will be continued herein.
As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under hybridization conditions, preferentially to target nucleic acid sequences. The term "preferentially" is used in a relative sense; one hybridization reaction product is more stable than another under identical conditions.
In addition to their hybridization properties, probes also may contain certain constituents that pertain to their proper or optimal functioning under particular assay conditions. For example, probes may be modified to improve their resistance to nuclease degradation (e.g. by end capping), to carry detection ligands (e.g. fluorescein, biotin, and avidin), to facilitate direct or indirect detection (.sup.32 P, and fluorescent and chemiluminescent agents) or to facilitate their capture onto a solid support (e.g., homopolymer "tails"). Such modifications are elaborations on the basic probe function which is its ability to usefully discriminate between target and non-target organisms in a hybridization assay.
Hybridization traditionally is understood as the process by which two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion (one oriented 5' to 3', the other 3' to 5') to form a double-stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules pertaining to which nucleic acid bases may pair with one another. The high specificity of probes relies on the low statistical probability of unique sequences occurring at random as dictated by the multiplicative product of their individual probabilities. Normal hybridization conditions for nucleic acid of approximately 10 to 250 nucleotides would include a temperature of approximately 60.degree. C. in the presence of 1.08M sodium chloride, 60 mM sodium phosphate and 6 mM ethylenediamine tetraacetic acid (pH 7.4).
Hybridization conditions are easily modified to suit nucleic acids of differing sequences. Factors which may influence the hybridization conditions for a particular nucleic acid composition are base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids.
Reaction parameters which are commonly adjusted are the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and the temperature of hybridization. Generally, as hybridization conditions become more stringent, or less favorable for hybridization, longer probes are required to form stable hybrids.